Date of Award
Doctor of Philosophy
Chemical reagents are an important tool in the study of biological systems. In this thesis some applications of chemical reagents in the study of biomolecules are presented. Selective cleavage of proteins is a common procedure in many biochemical applications ranging from standard protein sequencing to novel methods in proteomics and bioengineering. Pt(II) complexes are synthetic proteases, with the same selectivity as cyanogen bromide, they cleave at the C-terminus of methionine residues. Pt(II) reagent has several advantages over cyanogen bromide. For example, cyanogen bromide irreversibly converts methionine residues to serine lactone in 70% formic acid, while Pt(II) reagents perform hydrolytic cleavage in weakly acidic solutions. Cleavage of peptides and proteins by Pt(II) reagent is accelerated when performed under microwave irradiation, without any detected secondary reactions, and with the same selectivity as in the absence of microwaves. The study of RNA-protein interactions is another important area where chemical reagents are used, for example to probe conformational changes of RNA. Ribonucloprotein particles (RNPs) like the small subunit of the prokaryotic ribosome, our model system, are important components of biological systems. The small subunit of the ribosome (30S subunit) composed from a large ribosomal RNA (16S rRNA) and 21 ribosomal proteins (r-proteins) self-assembles in vitro. Temperature plays an important role in the assembly of the 30S subunit and one of the studies presented in this dissertation analyzes the importance of temperature in the interaction of a subset of r-proteins with 16S rRNA. R-protein/16S rRNA minimal RNPs are analyzed by base specific probing, and the significance of their differential temperature dependent behavior is discussed in the context of 30S subunit and RNP assembly;Directed hydroxyl radical probing from unique positions on r-proteins reveals the architecture of 16S rRNA around the probe. Fe(II)-derivatized S20 protein is used as a probe for the assembly of 30S subunit. Protein dependent conformational changes of 16S rRNA around the probe, Fe(II)-S20, during assembly are assessed by probing RNPs of different complexities.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Dutcă, Laura-Mirela, "Chemical reagents in the study of biomolecules " (2006). Retrospective Theses and Dissertations. 1905.